Global impact of diet and temperature over aquaculture of Octopus vulgaris paralarvae from a transcriptomic approach.

Common octopus, Octopus vulgaris, is an economically important cephalopod species. However, its rearing under captivity is currently challenged by massive mortalities previous to their juvenile stage due to nutritional and environmental factors. Dissecting the genetic basis and regulatory mechanism behind this mortality requires genomic background knowledge. A transcriptomic sequencing of 10 dph octopus paralarvae from different experimental conditions was constructed via RNA-seq. A total of 613,767,530 raw reads were filtered and de novo assembled into 363,527 contigs of which 82,513 were annotated in UniProt carrying also their GO and KEGG information. Differential gene expression analysis was carried out on paralarvae reared under different diet regimes and temperatures, also including wild paralarvae. Genes related to lipid metabolism exhibited higher transcriptional levels in individuals whose diet includes crustacean zoeas, which had an impact over their development and immune response capability. High temperature induces acclimation processes at the time that increase metabolic demands and oxidative stress. Wild individuals show an expression profile unexpectedly similar to Artemia fed individuals. Proteomic results support the hypothesis revealed by transcriptional analysis. The comparative study of the O. vulgaris transcriptomic profiles allowed the identification of genes that deserve to be further studied as candidates for biomarkers of development and health. The results obtained here on the transcriptional variations of genes caused by diet and temperature will provide new perspectives in understanding the molecular mechanisms behind nutritional and temperature requirements of common octopus that will open new opportunities to deepen in paralarvae rearing requirements.

Oral immunostimulation of the oyster Ostrea edulis: Impacts on the parasite Bonamia ostreae.

Bioactive compounds were orally administered to the native European oyster Ostrea edulis to evaluate the immune response and the progression of infection of the protozoan parasite Bonamia ostreae. The immunostimulants lipopolysaccharide and zymosan directly administrated to the water column induced an increase in lysozyme activity and the percentage of granulocytes in naïve oysters over a period of 7 days. In another set of experiments, zymosan and curdlan were microencapsulated in alginate and also administered to the water column to naïve and B. ostreae infected O. edulis. Oyster mortality, prevalence and intensity of infection and several immune parameters were evaluated up to 28 days post-administration. Lysozyme activity, nitric oxide production and the expression of galectin, lysozyme and superoxide dismutase increased after 24 h in both infected and uninfected oysters. Zymosan immunostimulated oysters displayed a decrease in the prevalence of B. ostreae infection not attributed to mortalities but which could be associated to the enhanced ability of immunostimulants to evoke an enhanced immune response in the oysters and reduce infection.

Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 µm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 µm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes.

Morphological characterization and functional immune response of the carpet shell clam (Ruditapes decussatus) haemocytes after bacterial stimulation.

The morphology and functionality of Ruditapes decussatus haemocytes have been characterized by light microscopy and flow cytometry, leading to the identification of three different cellular subpopulations. Granulocytes were the largest cells, the hyalinocytes were smaller and contained fewer granules and the intermediate cells showed a size similar to hyalinocytes and a higher number of granules. The phagocytosis of different particles and the associated production of oxygen radicals were measured by flow cytometric methods. Granulocytes were the most active cells, followed by the intermediate cells and hyalinocytes. The effect of stimulation of haemocytes with lipopolysaccharide (LPS), with a heat inactivated bacterial mixture or with the infection of Vibrio splendidus on the cell viability and the expression of selected immune-related genes were studied. While significant low levels of damaged cells were registered in LPS-stimulated cells, the treatment with dead bacteria or V. splendidus reduced cell viability 1 h, 3 h and 6 h after treatment. The stimulation of haemocytes with LPS and dead bacteria induced changes in the expression of defender against cell death (DAD-1), thrombin, prosaposin, inhibitor of apoptosis (IAP), factor B and C3 complement component.

Functional and molecular immune response of Mediterranean mussel (Mytilus galloprovincialis) haemocytes against pathogen-associated molecular patterns and bacteria.

The effect of live bacteria (Micrococcus lysodeikticus and Vibrio anguillarum), and PAMPs (poly I:C, zymosan, LPS, LTA and CpG) on the production of intermediate toxic radicals (respiratory burst activity and production of nitric oxide) and mytilin B, myticin C and lysozyme gene expression was studied in vivo and in vitro. In vitro, bacteria were able to modulate the haemocytes' respiratory burst activity, being significantly increased after 6 h of incubation. The effect of pathogen-associated molecular patterns (PAMPs) was also studied. Zymosan produced an increase of the PMA-mediated response but an inhibition of the zymosan-mediated response. A significant increase of nitric oxide production was found at all the sampled time points (1, 3 and 6 h) in comparison with controls on both, the Gram-positive and Gram-negative bacteria. The in vivo responses measured on haemocytes after M. lysodeikticus injection were faster than those induced by V. anguillarum. However, V. anguillarum induced stronger in vitro effects. Mytilin B, myticin C and lysozyme in vitro gene expression, occurred at short times after infection. The maximum in vitro expression was detected 3 h post-infection. The differences between M. lysodeikticus and V. anguillarum in different measured parameters may suggest that different signalling pathways might be involved. Moreover, among all assayed PAMPs, LPS elicited the highest response.

Differentially expressed genes of the carpet shell clam Ruditapes decussatus against Perkinsus olseni.

Suppression-Subtractive Hybridization (SSH) was used to identify differentially expressed Ruditapes decussatus genes against the protozoan Perkinsus olseni infection. A forward and a reverse subtraction were carried out to identify up- and down-regulated genes in both haemocytes and gills of clams naturally infected with P. olseni. New genes, candidates for further investigation into the functional basis of resistance to pathogens, have been detected for the first time in the clam (R. decussatus). A total of 305 differentially expressed sequences were obtained, 221 of them in haemocytes and 84 in gills of infected clams. The number of ESTs with potential similarity with known genes was 97, 42 among them were related with immunity and stress related functions. The pattern of expression of the immune selected genes was studied by quantitative PCR with samples of naturally Perkinsus infected clams and compared with samples from an in vitro infection of clam haemocytes with Perkinsus zoospores. The maximum expression was found 1h post infection. The complete open reading frames of selected sequences (Rd adiponectin-C1q and Rd DAD-1) were determined. Our results provide new insights into the molecular basis of host-pathogen interactions in R. decussatus.

Characterization of a C3 and a factor B-like in the carpet-shell clam, Ruditapes decussatus.

The alternative pathway is considered to be the most ancient route for activation of the complement system. Herein, we report the characterization of C3 and factor B-like proteins in the clam Ruditapes decussatus, termed Rd-C3 and Rd-Bf-like. The Rd-C3 is a three-chain protein, similar to other protoC3 proteins, and the Rd-Bf-like is composed of two complement control protein modules (CCP domains) that differ from other described Bf proteins. The inoculation of clams with live bacteria did not result in induction of these functions, but inhibited the expression of Rd-C3 and Rd-Bf-like.